Autoimmune Antibody Testing

Points of Note:
• The interpretation of all autoantibody tests is highly dependent on
the likelihood of disease in the patient.
• The results should always be interpreted with the clinical features of
the patient and never in isolation.
• Autoantibodies may be present in healthy individuals and may also
occur transiently with intercurrent illness or may be induced by drug
therapy. Conversely, autoimmune disease may be present in the
absence of detectable autoantibodies.
Do not use these tests as ‘screens’ for autoimmune disease but rather
decide the clinical diagnosis and the likelihood of autoimmune disease and
use specific autoantibody tests as diagnostic aids.

Anti-Nuclear Antibodies (ANA). (Incorporating Anti-double stranded

DNA (dsDNA) and Anti-Extractable Nuclear Antigen (ENA) Antibodies)

These tests are predominantly used for the investigation and diagnosis of
inflammatory connective tissue diseases such as SLE, Sjogren’s syndrome,
and systemic sclerosis, mixed connective tissue disease, polymyositis and
dermatomyositis.

An ANA panel may be positive in healthy individuals (and particularly with

increased age) or be induced transiently during acute illness or with infection,
and by certain medication. It may also be positive in many other autoimmune
diseases including rheumatoid arthritis and autoimmune thyroid disease. The
ANA has no particular clinical significance in these situations. Thus the tests
should only be requested where the clinical features are suggestive of
inflammatory connective tissue disease. Occasionally the ANA may be
negative where there is underlying CTD.

We currently use an automated machine (Bioplex) to detect antibodies to a

panel of antigens relevant to inflammatory connective tissue diseases. These
are detailed below. Like any assay, patients with disease may have negative
tests (false negatives) and patients without disease may have positive tests
(false positives). Low level positive tests may be non-specific in some
individuals and test results should always be interpreted with the clinical
features. In patients with CTD, tests may be positive before the patient
develops the full clinical features of the disease.
The general disease associations with these autoantibodies are as follows:

Anti-dsDNA Typically associated with SLE but high levels may

antibodies be found in autoimmune hepatitis. When positive,
the laboratory may check the result by performing a
second test (Crithidia assay) to confirm the
specificity of the result.
Anti-SSA (Ro) Associated with Subacute Cutaneous Lupus
Antibodies. Erythematosus, Systemic Lupus Erythematosus
(Ro60 and 52) and Sjogren’s Syndrome and are of particular
relevance during pregnancy as their presence may
be associated with neonatal lupus and congenital
heart block.
Antibodies to Ro52 without anti-Ro60 may be
associated with polymyositis.
Anti-SSB (La) Generally are present with anti-SSA antibodies and
Antibodies are associated with SLE and Sjogren’s Syndrome.
Anti-Sm Pathognomonic of SLE but only occur in a minority
Antibodies of patients with this condition.
Anti-Ribonucleolar Particularly associated with SLE or Mixed
protein (RNP) Connective Tissue Disease.
Antibodies
(SmRNP, RNP68)
Anti-Scl-70 Particularly associated with diffuse systemic
Antibodies sclerosis (scleroderma). A positive result may be
followed by analysing by a second method to
confirm specificity.
Anti-Jo 1 Found in a minority of patients with polymyositis,
Antibodies particularly when it is associated with interstitial lung
disease.
Anti-Centomere Typically associated with systemic sclerosis /
Protein B CREST Syndrome but also found in Primary Biliary
Antibodies Cirrhosis.
Anti-Chromatin May be associated with SLE (and other CTDs) but
antibodies usually in associated with the presence of other
autoantibodies. The relevance of low level anti-
chromatin antibodies in isolation is unclear.
Anti-Ribosomal P Appear to be highly specific for SLE although is also
antibodies described in some cases of autoimmune hepatitis.
May occur in isolation without other autoantibodies.
Rheumatoid Factor

This may be present in Rheumatoid arthritis but also in patients with Sjogren’s
syndrome, SLE or cryoglobulinaemia.
It is detectable in 15% of the population without RA following chronic
inflammation or infection or in the elderly.
RF may be negative in 15-30% of patients with adult RA.

Anti-Cyclic Citrullinated Peptide (CCP) Antibodies

These antibodies appear to be highly specific for Rheumatoid Arthritis and
their presence may be associated with erosive joint disease. Anti-CCP is
present in about 60-70% of patients with RA. There is no general correlation
with presence of RF. Monitoring anti-CCP levels is not useful during treatment
of RA.
The clinical utility of this test is generally limited to specialist Rheumatology
clinics.

Anti-Neutrophil Cytoplasmic Antibodies (ANCA) Antibodies

The ANCA assays are performed first by indirect immunofluorescence

providing a negative or positive result. Positive results are reported as a
pattern: P(erinuclear), C(ytoplasmic) or Nuclear. The presence of a positive
ANCA result is not disease-defining. A negative test does not exclude
vasculitis.

The disease association (small vessel vasculitis) is with specific antibodies

directed to one of two major granule proteins i.e. Myeloperoxidase (MPO) or
Proteinase 3 (PR3). All positive results are referred on for testing on the
Bioplex machine for the presence of IgG to these proteins. Tests for
autoantibodies to other neutrophil granule proteins are not available. A
negative result will generally be reported without further tests but very
occasionally anti-MPO or anti-PR3 antibodies may be present with a negative
ANCA.

If the clinical features are strongly suggestive of small vessel vasculitis and
the ANCA is negative then these tests may be undertaken on request.
In combination ANCA and anti-MPO and PR3 antibodies are about 90%
sensitive in detecting small vessel vasculitis. Thus a negative test does not
exclude vasculitis.

In general in the context of small vessel vasculitis:

C-ANCA is associated with anti-PR3 antibodies and is found in Wegener’s
Granulomatosis (Granulomatosis with Polyangiitis), and Churg-Strauss
Syndrome (Eosinophilic Granulomatosis with Polyangiitis).
P-ANCA is associated with anti-MPO antibodies and is found in Microscopic
Polyangiitis, Crescentic Glomerulonephritis and Churg-Strauss Syndrome.
This is variable and occasionally one may have C-ANCA with anti-MPO and
P-ANCA with anti-PR3, or combinations of antibodies. A positive ANCA with
negative anti-MPO and PR3 may be found in a variety of conditions including
autoimmune hepatitis, sclerosing cholangitis, ulcerative colitis, SLE, RA,
malignancy, cystic fibrosis and chronic infections. ANCA testing is generally
not warranted for these clinical conditions.

Anti-Glomerular Basement Membrane (GBM) Antibodies

Antibodies to GBM are primarily directed towards the non-collagenous domain

of the alpha 3 chain of type IV collagen. Since this type of collagen is found
predominantly in glomeruli and alveoli, the presence these directly pathogenic
antibodies is associated with rapidly progressive glomerulonephritis and
alveolitis (Goodpasture’s Syndrome).
Anti-GBM antibodies may also be found in some patients with ANCA positive
small vessel vasculitis, usually with anti-MPO antibodies.
Repeat tests are useful to determine the effectiveness of plasma exchange.

These tests are performed together as a ‘package’ on the Bioplex machine

and the result is reported as anti-PR3, anti-MPO and anti-GBM antibodies
together, even if only one of the autoantibodies is relevant.

Anti-Cardiolipin Antibodies

These tests are utilized to assist in the clinical diagnosis of the Anti-
Phospholipid Syndrome (APLS). This condition is characterized by vascular
thrombosis and/or recurrent fetal loss. Other features may include livedo
reticularis, thrombocytopaenia, heart valve disease, nephropathy and
neurological disease. APLS may be Primary, occurring alone, or Secondary,
associated with connective tissue disease, especially SLE.
Anti-Cardiolipin (aCL) antibodies of IgG and/or IgM isotype in serum or
plasma, present on medium or high titre (> 40 GPL or MPL), on two or more
occasions, at least 12 weeks apart, measured by standardized ELISA
Anti-β 2 glycoprotein-I antibody of IgG and/or IgM isotype on serum or plasma
(in titre > 99th percentile), present on two or more occasions, at least 12
weeks apart, measured by standardized ELISA, according to recommended
protocols

If this diagnosis is strongly suspected then anti-cardiolipin antibodies should

be requested. Samples for Anti-β 2 glycoprotein-I antibodies may be sent
away, after clinical discussion with the laboratory, if the test is negative and
clinical suspicion is high. In addition, aCL antibodies may be weakly positive
or transiently abnormal in numerous other conditions, a positive result needs
to be confirmed with a repeat sample at least 12 weeks later.

Tissue Specific Autoantibodies

This section refers to anti-gastric parietal cell, anti-mitochondrial, anti-smooth

muscle, and anti-liver kidney microsomal antibodies.
These tests are performed by indirect immunofluorescence, and all these
antibodies can be detected on a single slide.
Ideally the specific antibody relevant to the clinical features under
investigation should be requested.
As with all autoantibody testing these may be positive in healthy individuals
and may be non-specific.
Thus the tests should only be requested with specific reference to the clinical
condition.
In general the value of these tests is limited to the investigation of pernicious
anaemia and autoimmune liver disease.

Anti-Gastric Parietal Cell Antibodies & Anti-Intrinsic Factor antibodies

These are present in individuals with autoimmune gastritis and pernicious
anaemia. However they are not specific for these conditions, as they may be
also found in healthy individuals, particularly with increased age, and in those
with other autoimmune conditions (thyroiditis, Addison’s disease, IDDM).
If Vitamin B12 levels are low then anti-intrinsic factor antibodies should be
requested.
Anti-GPC antibodies are more sensitive, but less specific, for pernicious
anaemia than anti-IF antibodies.

Anti-Mitochondrial Antibodies
These antibodies are directed to pyruvate dehydrogenase complex and have
a close association with Primary Biliary Cirrhosis.
When these are positive for the first time then the sample will be tested for
Anti-PDH E2 (M2) antibodies to confirm the antibody specificity.

Anti-Smooth Muscle Antibodies

These are frequently non-specific or transiently detected.
However they may be associated with Type 1 Autoimmune Hepatitis.
These patients may also have a positive ANA.

Anti-Liver Kidney Microsomal Antibodies

These are directed against Cytochrome P450 proteins and are particularly
associated with Type 2 Autoimmune Hepatitis.
This form of AIH presents more commonly in children.

Anti-Thyroid peroxidase (TPO) and anti-TSH receptor antibodies.

Anti-TPO antibodies at high titre are found in patients with Hashimoto's

thyroiditis (95%), primary myxoedema (90%) and Graves disease (18%).
Low titre anti-TPO antibodies may occur in goitre, thyroid carcinoma and in
other organ-specific autoimmune diseases e.g. PA. The main value of this test
is in patients with borderline or compensated hypothyroidism. Strong positive
anti-TPO antibodies are predictive of progression to permanent
hypothyroidism.
Hyperthyroidism in Graves’ disease is due to autoantibodies to the TSH
receptor (TSHR) and measurement of these autoantibodies can be useful in
disease diagnosis and management.
Gluten Sensitive Enteropathy (Coeliac Disease) Testing

Coeliac Disease is an enteropathy that occurs in the presence of gluten found

in wheat (gliadins), barley (hordeins) and rye (secalins).
The condition is thought to be far more prevalent than previously realized and
may manifest with non-specific and extra-intestinal symptoms (anaemia,
osteopaenia, fatigue, abnormal liver function tests).
The enteropathy is essentially an autoimmune condition that occurs in the
presence of gluten and resolves with gluten withdrawal. IgA antibodies to
gliadin, tissue transglutaminase and to antigens created by the combination of
the two are involved in the disease process. GSE predominantly occurs in
those individuals with a specific tissue type, HLA DQ2.

Like all autoantibody tests, the results should be interpreted with the
clinical features.
The tests will not be positive in all patients with GSE, and some patients with
positive tests may not have GSE.
For accurate diagnosis the tests should only be undertaken with the
patient eating a normal gluten-containing diet for at least 6 weeks.
Those on gluten-free diets may have false negative tests.
The gold standard for the diagnosis of Gluten Sensitive Enteropathy is small
intestinal biopsy which should also be undertaken on a normal diet containing
gluten
In most patients positive tests alone are inadequate to make the diagnosis.
Follow up tests may be useful after the diagnosis has been made to follow
compliance with a gluten-free diet.
IgA deficiency, which occurs in about 1 / 400-700, will lead to false negative
serological tests for GSE. Using the results of the anti-TTG tests, we are able
to determine which samples to test for IgA levels. Where IgA deficiency is
present then IgG anti-TTG testing will follow. These are far less specific for
GSE and may be present in healthy individuals and those with other intestinal
diseases.

IgA Anti-Tissue Transglutaminase (tTG) Antibodies

The antigen within the endomysium to which the IgA antibodies bind in GSE
has been identified as tissue transglutaminase (tTG).
This is the main assay for testing for Coeliac Disease.

Anti-Endomysial Antibodies (EMA)

IgA anti-endomysial antibodies are detected by indirect immunofluorescence.
The results are reported as positive or negative. This assay will only be
performed if the anti-TTG result falls within the borderline range.
Special Autoantibody Tests

Anti-Saccharomyces cerevisiae Antibodies

Anti Saccharomyces antibodies (ASCA) are associated with Crohn’s disease
(CD) and are found in 50-60% of patients with CD and 10-15% of patients
with Ulcerative colitis (UC).
Combined IgG and IgA ASCA can give better discrimination between CD and
UC.
ASCA testing can be used to distinguish between CD and UC especially in a
Gastro-enterology clinic setting.

Anti-Striated Muscle Antibodies

These are detected by indirect immunofluorescence using skeletal muscle.
Testing for these antibodies is of most relevance in patients with myasthenia
gravis where their presence is associated with underlying thymoma.

Anti-Skin Antibodies
Skin antibodies are assayed by indirect immunofluorescence.
This test is used to aid in the diagnosis of autoimmune bullous skin disease
(Bullous Pemphigoid, Pemphigus vulgaris), particularly when direct
immunofluorescence on skin biopsy is unavailable.

Anti-Adrenal Cortex Antibodies

These are detected by indirect immunofluorescence using adrenal tissue.
This test is used to aid the diagnosis of autoimmune adrenal failure (Addison’s
disease).

Anti-Islet Cell Antibodies

These antibodies are used to assist in the diagnosis of type 1 (autoimmune)
diabetes.
The tests are usually positive early in the clinical course.
Additional tests (anti-IA2 and anti-GAD65) may be sent away to an outside
reference laboratory if necessary.

NICE Guidance (2015) states that "With autoantibody testing, carrying out
tests for 2 different diabetes-specific autoantibodies, with at least 1 being
positive, reduces the false negative rate”.
 Neurological Disease Antibodies
These are expensive tests and are referred to specialist laboratories
Acetylcholine Receptor (anti- Myasthenia Gravis:
AChR) IgG, detected by RIA Generalised 85%
Ocular 50%
MuSK (anti-MuSK) Generalised AChR antibody negative up to 50%
IgG, detected by RIA Myasthenia Gravis (15% of all MG patients of AChR neg
approx) MG (variable)
Voltage gated Ca2+ channel Lambert-Eaton Syndrome (with or without >85%
(anti-VGCC) IgG, detected SCLC) Around 30%
by RIA Cerebellar ataxia ass with SCLC
Voltage gated K+ channel Acquired neuromyotonia 40%
(anti-VGKC) IgG, detected Limbic encephalitis-like syndromes Not known
by RIA (both sometimes associated with thymoma
or SCLC)
Ganglioside (GM1) (anti-GM1) Guillain Barre Syndrome (IgG) ~40%
IgG and IgM (combined), Multifocal motor neuropathy (IgM) ~60%
detected by ELISA
Ganglioside (GQ1b) (anti- Miller-Fisher syndrome (IgG) >90%
GQ1b) Chronic sensory neuropathy (IgM) Some
IgG and IgM (combined),
detected by ELISA
Glutamic acid decarboxylase High levels >300 U/ml in Stiff-man syndrome ~60%
(GAD) (anti GAD) Cerebellar ataxia (usually with other Not known
IgG, detected by RIA autoimmune disorders)
Low levels <100 U/ml in Diabetes
Myelin associated Chronic sensory neuropathies Some
glycoprotein (MAG) (anti-
MAG)
IgM, detected by ELISA
Markers for Paraneoplastic Antigen: most common presentation (most Variable
neurological syndromes frequent associated tumour)
Anti-neuronal antibodies Hu, ANNA: Subacute sensory neuropathy/
detected by encephalitis (SCLC)
immunohistochemistry (Hu, Yo, Yo, APCA1: Cerebellar degeneration
Ri, Ma, Tr and potentially other (breast, ovary)
autoantibodies); if positive, Ri, ANNA2: Opsoclonus/Myoclonus and
followed by confirmation using other (breast)
immunoblotting on RAVO kit Ma2: Limbic encephalitis and other
syndromes (testicular and other cancers)
RAVO kit Amphiphysin: Opsoclonus, ataxia (breast,
Immunoblotting detects Hu, Yo, SCLC)
Ri, Ma, Amphiphysin, CRMP/CV2: Various (various)
CRMP/CV2 but not Tr or other Tr: Cerebellar ataxia (lymphomas)
antibodies)